Heroin and tapentadol promote accelerated senescence of SH-SY5Y human neuroblastoma cells at subtoxic concentrations

Background: Opioids are widely used in clinical practice due to their analgesic efficacy. However, their potential for abuse and deleterious effects represents a major global health concern [1]. Importantly, there is growing evidence suggesting that long-term opioid use may promote inflammatory responses and oxidative stress, altering molecular pathways involved in cell senescence, a feature often associated with accelerated ageing and cellular functional decline [2,3]. Objective: This work aimed to elucidate the impact of both recreationally and therapeutically used opioids (i.e., heroin and tapentadol, respectively) on neuronal cell ageing. Methods: SH-SY5Y human neuroblastoma cells were exposed to 1 nM and 1 µM of either heroin or tapentadol for 72h, and cell senescence was evaluated by measuring b-galactosidase activity (a widely used marker of senescent cells) using a commercially available kit (Abcam, USA). At the same time point, reactive oxygen species (ROS) levels were assessed using dichlorofluorescein (DCFH-DA). Notably, we have previously shown these opioids’ concentrations to be below toxicity thresholds. 25 nM doxorubicin was used as a positive control. In parallel, SH-SY5Y cells were chronically exposed, every 2-3 days, for a total of 30 days (between passages 21 and 25), to the same opioid concentrations. Genomic DNA was collected every other passage, and relative telomere length was measured through quantitative real-time PCR. Results: We observed increased b-galactosidase activity in opioid-exposed cells compared with untreated controls, in a concentration-dependent manner. Specifically, heroin increased this enzyme’s activity by 1.25- and 1.33-fold (for 1 nM and 1 µM, respectively), while tapentadol increased it by 1.19- and 1.29-fold at 1 nM and 1 µM, respectively. None of the opioids, at the tested concentrations, altered ROS levels after 72h exposure. From passages 21 to 25, telomere length decreased by 6% in control cells. However, opioid treatment exacerbated progressive telomere shortening, with reductions of 15.0 and 28.5% for tapentadol, and 10.0 and 27.1% for heroin at 1 nM and 1 µM, respectively. Conclusions: Overall, our preliminary data indicate that opioid use promotes early signs of cellular senescence and accelerated ageing (i.e., telomere shortening) in SH-SY5Y human neuroblastoma cells. Nonetheless, additional assays are ongoing to further characterize the effects of these opioids’ chronic exposure on neuronal cells.